Project Summary/Abstract In healthy individuals, immune responses are resolved in part by Foxp3+ regulatory T-cells (Treg). In individuals afflicted with type 1 diabetes (T1D) or Multiple Sclerosis (MS), Treg function well ex vivo but fail to prevent disease. This implicates in vivo impairments in Treg function in autoimmunity. A critical factor that governs Treg homeostasis is the cytokine interleukin 2 (IL-2). Unlike some cytokines that circulate freely, IL-2 is mostly bound to the ECM, specifically to heparan sulfate (HS). We have identified a heretofore ignored, fundamental role for HS-mediated IL-2 sequestration in Treg function and stability in vivo. It is well-established that HS-containing proteoglycans (HSPGs) retain and slowly release other cytokines and growth factors over time, thereby contributing to tissue remodeling after injury. We find that inflammation increases the ability of tissues to retain IL-2. We also find that HS-bound IL-2 (HS/IL-2) functions as a superagonist of IL-2R signaling, promoting the expansion and stability of Treg. Finally, we find that heparanase (HPSE) and HS catabolism are required for Treg function in vitro and in vivo, suggesting that Treg may use HPSE to strip IL-2 from HSPG within the extracellular matrix. Conversely, HS/ IL-2 sequestration and acquisition may be impaired in autoimmunity. HS is abundant in healthy pancreatic islets but disappears in the setting of autoimmune insulitis. Similarly, patterns of HSPGs are deranged within white matter lesions in MS. It may be possible to recapitulate HS/IL-2 signals therapeutically. Capitalizing on the tolerogenic properties of HS/IL-2, we have developed synthetic mimetics of HS/IL-2 that expand Treg. Our vision is that these can be conjugated to antigens and used to induce Treg and prevent MS and T1D. In light of these exciting preliminary data, we hypothesize that HS/ IL-2 promotes Treg function and that this pathway is impaired in autoimmunity. We will test this hypothesis in experiments with the following aims: In Aim 1 we will evaluate ECM binding of cytokines in T1D and MS. In Aim 2 we will elucidate how HS/IL-2 acts as a superagonist of IL-2R signaling. In Aim 3 we will develop HS/IL-2 complexes that promote Treg induction and immune tolerance. Together these Aims have the potential to transform our understanding of the mechanisms that promote immune homeostasis in peripheral tissues.